Epigenetic role of LINE-1 methylation and key genes in pregnancy maintenance

Spontaneous abortion is a pregnancy complication characterized by complex and multifactorial etiology. About 5% of childbearing women are globally affected by early pregnancy loss (EPL) and most of them experience recurrence (RPL). Epigenetic mechanisms and controlled inflammation are crucial for pregnancy maintenance and genetic predispositions may increase the risk affecting the maternal–fetal crosstalk. Combined analyses of global methylation, inflammation and inherited predispositions may contribute to define pregnancy loss etiopathogenesis. LINE-1 epigenetic regulation plays crucial roles during embryo implantation, and its hypomethylation has been associated with senescence and several complex diseases. By analysing a group of 230 women who have gone through pregnancy interruption and comparing those experiencing spontaneous EPL (n = 123; RPL, 54.5%) with a group of normal pregnant who underwent to voluntary interruption (VPI, n = 107), the single statistical analysis revealed significant lower (P < 0.00001) LINE-1 methylation and higher (P < 0.0001) mean cytokine levels (CKs: IL6, IL10, IL17A, IL23) in EPL. Genotyping of the following SNPs accounted for different EPL/RPL risk odds ratio: F13A1 rs5985 (OR = 0.24; 0.06–0.90); F13B rs6003 (OR = 0.23; 0.047–1.1); FGA rs6050 (OR = 0.58; 0.33–1.0); CRP rs2808635/rs876538 (OR = 0.15; 0.014–0.81); ABO rs657152 (OR = 0.48; 0.22–1.08); TP53 rs1042522 (OR = 0.54; 0.32–0.92); MTHFR rs1801133/rs1801131 (OR = 2.03; 1.2–3.47) and FGB rs1800790 (OR = 1.97; 1.01–3.87), although Bonferroni correction did not reach significant outputs. Principal Component Analysis (PCA) and logistic regression disclosed further SNPs positive/negative associations (e.g. APOE rs7412/rs429358; FGB rs1800790; CFH rs1061170) differently arranged and sorted in four significant PCs: PC1 (F13A, methylation, CKs); PC3 (CRP, MTHFR, age, methylation); PC4 (F13B, FGA, FGB, APOE, TP53, age, methylation); PC6 (F13A, CFH, ABO, MTHFR, TP53, age), yielding further statistical power to the association models. In detail, positive EPL risk association was with PC1 (OR = 1.81; 1.33–2.45; P < 0.0001) and negative associations with PC3 (OR = 0.489; 0.37–0.66; P < 0.0001); PC4 (OR = 0.72; 0.55–0.94; P = 0.018) and PC6 (OR = 0.61; 0.46–0.81; P = 0.001). Moreover, significant inverse associations were detected between methylation and CKs levels in the whole group (rIL10 = − 0.22; rIL17A = − 0.25; rIL23 = − 0.19; rIL6 = − 0.22), and methylation with age in the whole group, EPL and RPL subgroups (r2TOT = 0.147; r2EPL = 0.136; r2 RPL = 0.248), while VPI controls lost significance (r2VPI = 0.011). This study provides a valuable multilayer approach for investigating epigenetic abnormalities in pregnancy loss suggesting genetic-driven dysregulations and anomalous epigenetic mechanisms potentially mediated by LINE-1 hypomethylation. Women with unexplained EPL might benefit of such investigations, providing new insights for predicting the pregnancy outcome and for treating at risk women with novel targeted epidrugs.

Moreover, genetics and epigenetics interplay is of interest for both those genes directly involved in the methylation processes, and those related to inflammation, immunity, angiogenesis and blood group, widely explored by association studies and meta-analyses suggestive of the existence for inheritance traits.
To investigate whether phenotypic variability in miscarriage was under any hereditary influence, a large study explored genetic and environmental influences on miscarriage rates by a twin study 39 .The authors, by analyzing 3234 female twins equally distributed between monozygotic and dizygotic did not find genetic variation (heritability) as a common cause of miscarriage apart from abnormal embryo karyotype, concluding that women propensity to miscarriage has low heritable basis, though genetic effects might be maintained by a constant and common insertion of novel mutations.On the other hand, single or compound analyses of gene variants support a genetic predisposition in the mother and or in the embryo, and also considering the case of the vanished twin or of the reduced fetal viability for particular coinheritance in cis or trans of MTHFR gene variants, this concept has not been confirmed by other studies that concluded the dizygotic twinning is not associated with MTHFR haplotypes [40][41][42] .On the other hand, a recent report on the genetic architecture of sporadic and multiple miscarriage concludes stating that its complex etiopathogenesis is driven in part by genetic variations mainly related to placental biology, adding that SNP-heritability together with other acquired circumstances may have a larger contribution 43 .Finally, several recent investigations on selected groups of SNPs recognized promising pathways to be further explored.
MTHFR gene (1p36.22) encodes a pivotal enzyme in cycling folate isoforms, producing the most active methyl-THF (CH3-THF).MTHFR gene variants (i.e., rs1801133, C677T and rs1801131, A1298C) significantly affect the enzyme activity leading to intracellular folate unbalancing affecting the maternal-fetal crosstalk during pregnancy as hypothesized in the GEMCDS 12,13,44 .Association of homocysteine, CRP, fibrinogen and gestational diabetes with EPL and poor pregnancy outcomes have been recently published 45 .
CRP gene (1q23.2) encodes a protein involved in the complement cascade activation and amplification.CRP greatly increases during the acute phase or other inflammatory stimuli and is associated with host defense based on its ability to recognize foreign antigens and damaged cells by interacting with humoral and cellular effector systems.High levels are associated to preterm delivery and other complications 46 and CRP genotype and maternal plasma levels in the first trimester have been also investigated 47 including rs2808635/rs876538 variants being associated to basal and stimulated CRP levels 48 .
FGA and FGB genes belong to the fibrinogen cluster (4q32.1-4q31.3),FGG gene included.Stimulated by proinflammatory triggers also cooperate in the quality of the 3D-organization of the fibrin scaffold necessary in every heling process and also crucial in the blastocyst implantation and embryo transfer after IVF 49,50 .Associations of gene variants in FGA (rs6050) and FGB (rs1800790) with pregnancy outcome have been investigated connecting both fibrinogen levels and fibrin architectures 51 .
F13A1 (6p25.1)and F13B (1q31.3)genes have been investigated in complex diseases and in pregnancy loss with controversial results in association or not with fibrinogen levels often considering their main functional gene variants coinherited with the fibrinogen gene cluster polymorphisms [51][52][53] .Combined investigations merit particular importance because rs5985 and rs6003, respectively in F13A1 and F13B genes, interact with the activation of the FXIIIA2B2 complex and with the fibrinogen gene cluster in both acute inflammation and coagulation phases tuning in turn fibrinogen levels, 3D-fibrin architecture and MMPs resistance in any healing or remodeling phases 35,[54][55][56][57] .
TP53 gene (17p13.1)has been widely investigated as tumor suppressor favoring genome stability.Its role in reproductive medicine and placental vasculature has been recently investigated, since many of the steps involved in implantation-apoptosis rate are regulated by p53, moreover the key gene variant P72R (rs1042522) has been studied in pregnancy maintenance, including implantation failure, IVF, and prenatal sex selection [58][59][60][61][62] .
CFH gene (1q31.3)has essential role in the complement cascade activation and regulation also crucial for placentation and fetus development 63 .Among the several SNPs of complement cascade genes investigated in pregnancy complications 64 , rs1061170 has been found associated with RPL by uric acid and triglyceride anomalous levels during pregnancy 65 .
APOE gene (19q13.32)and Ɛ4 allele/haplotype (rs7412/rs429358) have been mostly investigated in neurodegeneration and cognitive impairment and recently in the quality of immune response after SARS-CoV2 vaccine [66][67][68] .Its role in RPL and implantation failure has been investigated as single gene or in combination with selected candidate genes by meta-analysis ascribing to the Ɛ4 allele the highest risk 69,70 .
ABO gene (9q34.2) by the association with ABO blood group has been considered an independent risk factor in the occurrence of pregnancy related complications with different results 71,72 .Interestingly, ovarian capacity and menstrual disorders have been associated to blood groups and the common ABO gene variant (rs657152) may be an interesting candidate in the maternal tolerance-rejection processes 73,74 .
Finally, pro-and anti-inflammatory CKs have been largely explored in pregnancy due to their role in the finely regulated immune programming 75 aimed at conferring the required tolerance to the developing embryo and the appropriate protection against pathogens 76,77 .As a consequence, placental immunology dysfunctions and/or disturbances in specific subsets of maternal immune cells (e.g., natural killer, NK), T-helper unbalance (Th1/Th2) or between regulatory T cells (Treg) 78 and Th17 are involved in EPL [79][80][81] .A realistic vision of CKs role is that they do not play independent pathogenic actions, rather they are part of a more complex regulatory network methylation score included 25,82 .
The main aim of the present research is to improve a mere case-control comparison performed by single gene analysis towards a more robust and multilayer tool able to disclose complex mutual interactions hardly recognizable by standard statistical approaches.For these reasons, we investigated the association between global DNA methylation and SNPs of selected genes involved in immune regulation, implantation-apoptosis, angiogenesis, genomic stability, together with selected inflammatory markers (i.e.CKs) as key factors affecting the risk of EPL

Study design and samples collection
A retrospective study aimed at assessing inherited/acquired predispositions to EPL has been performed in a cohort of 230 pregnant women by comparing those who experienced spontaneous miscarriage (EPL, n = 123) with a group of pregnant who underwent voluntary interruption according to the Italian law, 194, Art.6 comma b (VPI controls, n = 107) referring to the Hospital-University of Ferrara, Italy.The study involving human participants was reviewed and approved by the local regional ethical committee, the participants provided their written informed consent to participate in the study.The following exclusion criteria have been considered: (1) concomitant infections; (2) immune deficiency condition or immunosuppressive treatments; (3) inherited predispositions to abortion; (4) severe uterine malformation; (5) endocrine unbalancing and (6) accidental intake of teratogenic drugs.Finally, close relative patients have been excluded from the study.
Population characteristics for the whole group and after stratification by EPL-cases and VPI-controls are shown in Table 1.Participants had a gestational age ≤ 12 weeks and underwent to whole blood draw, plasma samples were processed within 2 h from drawing blood, and they were frozen at − 80 °C in multiple aliquots and blind tested.DNA was isolated from frozen whole blood by using an automated DNA extraction and purification robot (BioRobot EZ1 system, Qiagen; Hilden, Germany).

Inflammatory CKs analysis in plasma samples
Frozen plasma samples were analysed by the MILLIPLEX MAP Human Cytokine/Chemokine high sensitivity panel (Merck Millipore, Billerica, MA) to simultaneous quantify the following human CKs: IL6, IL10, IL17A, IL23.Samples were processed according to manufacturer's protocols and read on a MAGPIX instrument 86 equipped with the MILLIPLEX-Analyst Software using five-parameter nonlinear regression formula to compute CKs concentrations from the standard curves as previously described 87,88  Genotypes, methylation, CKs concentration and age were subjected to PCA.SNPs were scored 1, 2, 3 to represent common homozygous, heterozygous, and rare homozygous variant respectively, to indicate an increasing copy number of the variant allele (i.e., 0, 1, 2 respectively).As regards CRP and MTHFR variants, when not specified the gene symbols account for both variants analysed, unless otherwise specified (i.e., CRP(1) accounts for rs876538; CRP(2) accounts for rs2808635; MTHFR(1) accounts for rs1801133; MTHFR(2) accounts for rs1801131); finally, APOE (rs7412/rs429358) accounts for ε3/ε4 haplotypes as previously described 67 .Age, methylation and CKs were centred and scaled before PCA according to the formula (x-value-mean value)/SD; [Z = (x -μ)/σ].Collinearity diagnostic evaluation was assessed by variance inflation factor and values below 5.0 have been considered as threshold.PCA was performed by retaining those PCs with Eigenvalue exceeding 1.0.Eigenvector of independent variables with absolute value exceeding 0.3 (+ or −) was included.Variables with a loading above the cut-off point 0.3 were considered to be dominant in a component.Scores for each PC for each individual were extracted by using regression models.Retained PCs were computed in logistic regression analysis for presence/absence of EPL (Yes = 1, No = 0) versus PCs.P-values were two-sided with threshold for statistical significance fixed to P ≤ 0.05.

Ethics approval and consent to participate
This study was approved by the Ethics Committee of the Hospital-University of Ferrara, Italy (Protocol n. 91-2013, 13/11/2014; PRUA1GR-2013-00000220), samples were collected after the patient signed an informed consent form according to the Declaration of Helsinki, all relevant ethical regulations were followed.

LINE-1 DNA methylation
Population group and subgroups are as shown in Table 1.LINE-1 mean methylation in EPL was significantly lower than in VPI controls (81.34 ± 4.66 vs 85.82 ± 3.65; respectively; P < 0.00001).Intra case analysis ascribed to the RPL subgroup the lowest mean methylation level when compared to the remaining EPL subgroup (80.39 ± 4.29 vs 82.48 ± 4.87 respectively; P = 0.001).In addition, the different mean age in the cases and control group did not account for changes in the statistical significance of the comparison when the test was corrected for age (adjusted P-value = 0.0001).
As shown in Fig. 1a, a negative correlation existed between methylation levels and age, and it was stronger among the whole EPL cases than VPI controls (r 2 = 0.136 vs. r 2 = 0.011 respectively) as well among RPL subgroup when compared to the remaining EPL subgroup (r 2 = 0.248 vs. r 2 = 0.023 respectively) (Fig. 1b).Moreover, by stratifying the age-matched subgroups (EPL cases vs VPI controls) both the mean methylation level comparison and the age-methylation correlations kept trends in favor of the control group, confirming that regardless different mean age, EPL cases had significantly lower mean methylation levels (P = 0.0001) and higher age-dependent methylation declining (r 2 = 0.140 vs. r 2 = 0.033 respectively).
Interestingly, the same subanalysis (i.e.age vs methylation) stratified by MTHFR genotype, ascribed to the T-677 dysfunctional polymorphic allele a more robust negative correlation with age either by comparing the opposite genotypes in the whole cohort (r 2 = 0.123 vs r 2 = 0.212 respectively in 677C-carriers and 677TT-genotype; P = 0.025), or by comparing the TT-genotype in EPL versus VPI subgroup (r 2 = 0.283 vs r 2 = 0.057 respectively; P < 0.01) (Supplementary Fig. 1).

Single gene analyses
Table 2 shows the EPL risk calculation (crude OR and P-values) according to the genetic model applied and the considered subgroup of cases (see Supplementary Table 1 for the complete dataset).All the SNPs investigated were in Hardy-Weinberg equilibrium except for CFH rs1061170 (X 2 = 6.852) and ABO rs657152 (X 2 = 7.064).
F13A1 rs5985 genotypes were differently distributed between whole cases and controls (P = 0.069), reaching significant results in the alleles frequency comparison (P = 0.04).The under-representation of the T-allele in the case group yielded OR-values below the unit value for both genotype recessive model (OR = 0.24; 0.065-0.90;P = 0.03) and allele (OR = 0.60; 0.37-0.98;P = 0.04) comparisons.This accounted for a protective effect against EPL of more than 4-folds in TT-homozygous women.
F13B rs6003 genotypes were differently distributed between whole cases and controls (P = 0.07), reaching significant results in the alleles frequency comparison (P = 0.015).The under-representation of the C-allele in the case group yielded OR-values below the unit value for both genotype recessive model (OR = 0.23; 0.04-1.1;P = 0.07) and allele (OR = 0.50; 0.28-0.88;P = 0.015) comparisons.This latter accounted for a protective effect against EPL of about 2-folds in C-carrier women.The protective effect was even higher (i.e.3-folds) in the RPL subgroup (OR = 0.33; 0.13-0.87;P = 0.025).
FGA rs6050 genotypes were similarly distributed in the whole cases and controls, reaching borderline significant results by comparing the allele frequency in the RPL subgroup (OR = 0.58; 0.33-1.0;P = 0.05).
Vol:.( 1234567890 www.nature.com/scientificreports/FGB rs1800790 genotypes were similarly distributed in the whole cases and controls, though appreciable results were found in both allele (OR = 1.7; 0.98-2.98;P = 0.06) and genotype dominant model (OR = 1.97; 1.01-3.87;P = 0.045) comparisons in the younger subgroup of cases.This latter accounted for an increased EPL risk of about 2-folds in A-carrier women.
CRP rs2808635 genotypes were similarly distributed between whole cases and controls, though significant results were found in the younger subgroup of cases both in genotype distribution (P = 0.03) and recessive model  www.nature.com/scientificreports/(OR = 0.15; 0.014-0.87;P = 0.035) comparisons.This latter accounted for an increased EPL risk of more than 6-folds in G-carrier women.ABO rs657152 genotypes were similarly distributed between whole cases and controls (P = 0.08).Nonetheless, the under-representation of the TT-genotype in the case group yielded OR-values below the unit value in the genotype recessive model comparison (OR = 0.48; 0.22-1.08;P = 0.07).The protective effect was even higher in the RPL subgroup (OR = 0.23; 0.06-0.82;P = 0.02).This latter accounted for a protective effect against EPL of more than 4-folds in TT-homozygous women.
TP53 rs1042522 genotypes were similarly distributed between whole cases and control groups (P = 0.069).Nonetheless, the under-representation of the G-allele in the case group yielded OR-values below the unit value in the genotypes dominant model comparison (OR = 0.54; 0.32-0.92;P = 0.03).The protective effect was even higher in the younger subgroup of cases (OR = 0.44; 0.23-0.88;P = 0.02).This latter accounted for a protective effect against EPL of more than 2-folds in the G-allele carrier women.
EPL risk calculation (crude OR and P-values) for the above mentioned SNPs is summarized in Table 2, after Bonferroni correction they did not reach significant outputs.
The remaining SNPs in CFH (rs1061170) and in APOE (rs7412; rs429358) genes did not yield significant results by single analysis, but they were included in the PCA multilayer exploration.

Serum cytokine profile
Figure 2 shows the mean circulating levels of IL6, IL17A, IL23 and IL10 significantly higher in the whole case group than in the controls (P < 0.0001).By comparing RPL versus the remaining EPL cases no statistical differences have been observed (data not shown).
Interestingly, strong inverse correlations have been obtained between each single CK mean level and mean methylation level in the whole group of 230 pregnant women (Fig. 3).Correlation was lost in the RPL subgroup and retained in the remaining EPL cases.

PCA and logistic regression analysis of the principal components (PCs)
We performed PCA and logistic regression analysis to explore relationships between the significant PCs and the risk of EPL.PCA was completed with all the 18 variables and the first 7 PCs have been retained (i.e., eigenvalue > 1.0) explaining approximately 65.8% of the total variation.3D-loading plots show how all the 18 computed variables allocate (Fig. 4) and how the whole group of 230 cases stratifies (Fig. 5) along with the first three selected PCs overall explaining about 40% of dataset intergroup variance.

Discussion
Pregnancy loss is a challenging area of the reproductive medicine in which the maternal-fetal crosstalk initiates a series of complex biochemical and cellular interactions in large part genetically and epigenetically compelled [89][90][91] .According to the fetal origin of adult disease (FOAD), as well as the theory of the developmental origins of health and disease (DOHaD), maternal genetics and epigenetics burden have a great part.In utero and periconceptional exposures to environmental factors may act on genetic predispositions leading to pathological outcomes as pregnancy loss, and among the mechanisms linking environment and genetics, epigenetics (via methylome changes) plays a key role 16,17 .EPL pathogenesis is not fully understood lacking in large part a causative recognition.To increase knowledge in this field, our approach assessed epigenetic, genetic, and biochemical investigations in a well characterized cohort of 230 pregnant women by single analyses and multilayer PCA approaches.
In general, reduction of LINE-1 methylation can be linked to reduced methyl-donor availability via one-carbon metabolism 16 .High methyl groups availability does not necessarily result in increased LINE-1 methylation; Vol:.( 1234567890 www.nature.com/scientificreports/ a proper channelling of methyl groups in the DNA methylation path is directly dependent on DNMTs enzyme activities and the several one-carbon metabolism enzymes ultimately represented by MTHFR 44,92 .Both groups of genes are highly polymorphic and functional gene variants can significantly alter direct associations as recently demonstrated in complex phenotypes as cancer, maternal LINE-1 methylation in Down syndrome, and in type 2 diabetes patients with pre-symptomatic dementia [93][94][95] .All these mechanisms led to DNA damage and instability reducing faithful DNA synthesis and promoting cell senescence also favouring detrimental LINE-1s activation and in turn aberrant host gene expression.A part, drastic effect as embryo death, abnormal epigenome can influence the onset of infant complex diseases as paediatric cancers or neuro neurodevelopmental diseases in which genetic-epigenetic mother-child dyad has a role (GEMCDS-Study) 12,13 .
The main result of our study is a clear progressive global methylation reduction found in spontaneous miscarriages compared to normal pregnant controls, in which RPL cases also showed the lowest methylation levels when compared either to controls or single pregnancy loss cases.Furthermore, the age-matched subgroup comparison ascribed to cases a significantly stronger age-effect on the lowering grade of the methylation trend, further confirmed in the intra-cases suggesting a basic dysregulation of the epigenetic mechanisms essential for the maintenance of a healthy pregnancy 27,76 .In this line, correlations between epigenetic clocks and anti-Müllerian hormone or ovarian reserve or successfully IVF have been reported 27,96,97 suggesting that accelerated epigenetics mechanisms might determine the pregnancy outcome 98 .Abnormal in utero methylation setting may led to early embryo death, since DNA methylation greatly influences early embryo development and trophoblast proliferation assisting spiral artery remodelling essential for embryo implantation and maintenance of an effective maternal-foetal crosstalk 29 .Balanced de novo DNA methylation is not only critical during placentation but also for embryo survival as very recently demonstrated due to a critical role of DNMT3B action 99 .Accordingly, extensive maternal health during pregnancy, including a balanced methylation status and appropriate methyl groups availability, may have permanent impacts on the future health of descendants via global or specific epigenetic mechanisms.Noteworthy, a more robust negative correlation between age and methylation levels was observed in the MTHFR 677TT dysfunctional genotype, and at a greater extent in the spontaneous abortion subgroup supporting the concept that a suboptimal haplotype-driven intracellular methyl-groups availability exists 13,44 , and it may affect embryo survival and pregnancy maintenance.
DNA methylation of imprinted genes, and/or genes directly or not related to methyl groups cycling, uterine immune tolerance, inflammation, neo-angiogenesis, apoptosis, cytokine expression, and lipid or folate metabolism, globally contribute to the kaleidoscope of pregnancy maintenance 100 .On the other hand, many genetic risk factors have been largely investigated, mainly by SNPs analysis and meta-analyses utilized for risk prediction, often leading to conflicting or partial results 100 .
Among the most investigated variants there are those of the MTHFR gene (rs1801133, rs1801131), that play a key role in the availability of active folate isoforms essential for both faithful DNA neo-synthesis and balanced de-novo DNA methylation, crucial processes in embryo survival and foetal growth 12,101 .MTHFR T-677 allele (rs1801133) causes low intracellular level of 5CH3-THF, the most active isoform for methyl-group unit transfer by DNMTs.Moreover, a direct correlation has been found between global methylation and systemic inflammation assessed by high CRP levels particularly among carriers of the MTHFR T-677 allele that causes global    www.nature.com/scientificreports/hypomethylation in the low folate range and hyperhomocysteinemia also leading to incomplete vasculature and decreased placental transport 102,103 .On the other hand, CRP rs2808635 and rs876538 gene variants modulate the basal and stimulated circulating levels of CRP 48 , and several studies ascribed to these SNPs prognostic pharmacogenomics information on treatment and drug response included the extent of the humoral response after COVID-19 vaccination 67,[104][105][106] .CRP genotypes and CRP levels in pregnant women have been widely investigated 15,19,21 and rs2808635/rs876538 variants are associated to basal and stimulated circulating levels of CRP and inflammation.
Systemic inflammation reflects not only high circulating CRP and CKs levels but also fibrinogen concentration considered one of the reactive-phase molecules particularly important in pregnancy 107 .Fibrinogen is a target of autoimmune reactions and is involved in the generation of a stable 3D-fibrin meshwork necessary for inflammation control.Stability and perfect fibrin architecture also depend on coagulation FXIIIA 108,109 and complex fibrinogen/FXIIIA haplotypes (FGA, FGB, FGG, F13A1) have been found related to CRP levels during acute-phase reactions 110 .For these reasons, the most investigated SNPs (FGA rs6050, FGB rs1800790, FGG rs1049636, F13A1 rs5985 F13B rs6003) should be globally considered and analysed in complex diseases because of common functional (i.e., 3D-Fibrin structure organization) and genetic associations (i.e., Fibrinogen genes cluster; 4q32.1-4q31.3).Accordingly, compound haplotypes investigations might better account for the global risk assessment.
Similarly, F13A1 and F13B genes carry two main functional loci (rs5985, rs6003 respectively) synergistically involved in the catalytic enzyme activation of the FXIIIA2B2 tetramer, not only in the 3D-Fibrin meshwork organization together with FVII/TF complex 111,112 , but also in the novel angiogenesis processes and tissue healing 109,113 via TSP1-inhibition and VEGF expression 114 crucial mechanisms in embryo implantation and pregnancy maintenance [51][52][53]85 .
Normal embryo growth also needs appropriate trophoblast proliferation and adequate neo-vessel development; therefore, balanced angiogenesis and apoptosis play important roles for cyto-trophoblast development.TP53 coding product (i.e., p53 protein) gives protection to germinative cells and embryos by LIF-regulation a crucial cytokine helpful in blastocyst successful implantation 115 .A recent metanalysis on the role of TP53 rs1042522 found associated risk for RPL in women carrying the P72-allele in every genetic model analyzed 116 and among the recent explanations for this phenomena, reduced apoptosis, impaired placental structure lacking adequate gas and nutrient exchange, and a prolonged arrest of cells in G1-cycle have been proposed as mechanistic causative reasons 62 .
The role of maternal ABO blood group and pregnancy outcome have been extensively investigated and association with hypertension, preterm birth, diabetes, and cardiovascular complications are well known 117 .Recent attention has been done toward ABO rs657152 as responsible for maternal tolerance-rejection processes 73,74 suggesting involvement of the immune response in carrier cases as recently found in the dynamic of circulating antibody levels detected in healthy subjects after anti-SARS-CoV-2 vaccine 67 .
Basically, a single variable analysis just in part can explain the global complex mechanism responsible for EPL, and after Bonferroni correction for multiple comparisons the investigated SNPs did not reach significant outputs.Globally, the causative discussed rationale altogether reminds to unbalanced inflammation, angiogenesis, apoptosis, immunity, and methylation dysfunctions.In an explorative attempt we decided to investigate the same variables in a cumulative statistics approach accounted by PCA to have a more realistic and comprehensive picture as recently reported for inflammatory biomarker profiles and adverse birth outcome 82 also supported by the correlation we also found between inflammation (i.e., CKs levels) and methylation 25,118 .Interestingly, PCA analyses in the whole group yielded four principal components with different variables clustering, and these were also significantly associated with the risk of EPL as confirmed by further logistic regression analysis.In detail, PC1 mainly explains cytokines, methylation, and coagulation F13A1; PC3 is mainly represented by CRP, MTHFR, age and methylation.Finally, PC4 was strongly characterized by F13B, FGA, FGB and TP53, together with APOE, age and methylation, while PC6 was characterized by F13A, CFH, ABO, MTHFR, TP53 and age.With regard to PC2 and PC5, thought with borderline significant associations in regression analysis, they yielded interesting outputs: in PC2, CRP represented almost all of the component constituents with ABO and MTHFR, and PC5 was a comprehensive measure of F13B, FGA, MTHFR, and TP53.Considering the further PCA analysis performed by excluding those few cases in which cytokines have not been assessed, the outputs largely resembled those of the whole group and CRP accounted by PC2 reached now stronger significant association in logistic regression analysis.

Conclusion
The present explorative analysis suggests how a multilayer approach accounting for genetic, epigenetic, and biochemical factors may allow a rigorous EPL risk assessment.This is in line with the multifactorial nature of spontaneous EPL in which the coexistence of different factors may have effects on the final clinical phenotype showing additive/synergic/antagonistic effects finely detectable by multiple analysis procedures.Several questions have been raised, and to better answer them larger population cohorts must be recruited, also by investigating the foetus (epi)genome.Although the maternal (epi)genetics landscape has a protagonist role, the mother-foetal crosstalk is not less important as estimated by the GEMCDS group in other complex diseases in which the inutero origin of the disease has to be considered 12,13 .Recognizing the etiopathogenesis of EPL embraces great promise and will help to identify prognostic biomarkers and efficient therapeutic targets, as well as designing of novel epidrugs, inducing favorable epigenetic modulation to target and modulate the epigenetic pathways.

Figure 1 .Table 2 .
Figure 1.Methylation-age correlation analysis.(a) Correlation between methylation and age distribution in the whole cohort stratified by VPI controls (green dots) and EPL cases (red dots).Regression lines are shown (green line and red line for VPI controls and EPL respectively).(b) Correlation between methylation and age distribution in the EPL group stratified by single event (non-recurrent) cases (sEPL, grey dots) and recurrent EPL (RPL, dark dots).Regression lines are shown (continuous and dotted line for RPL and sEPL respectively).Each panel shows the r 2 -coefficient for the regression lines.

Figure 2 .
Figure 2. CKs levels distribution.Circulating IL6 (a), IL10 (b), IL17A (c) and IL23 (d) levels in VPI controls and EPL cases.Box plots show median and IQR.P values are indicated on top of each panel.

Figure 3 .
Figure 3. Pearson correlation heatmap between methylation and CKs in the whole group.Red and green indicate a positive and a negative association, respectively.Colour intensity represents the strength of the correlation.

Table 1 .
. Demographic and clinical data of EPL and VPI control groups.

cohort (n = 230) EPL cases (n = 123) VPI controls (n = 107)
Statistical analyses were performed using SPSS Statistics Version 22 (SPSS Inc., Chicago, IL, USA) and MedCalc version 20.112 (MedCalc Software Ltd., Ostend, Belgium).All figures were produced by GraphPad Prism9 (GraphPad Software, Inc., San Diego, California USA), unless otherwise specified.The Kolmogorov-Smirnov test was used to verify variables normal distribution.Normally distributed data are presented as mean and SD, while non-normally distributed data are presented as the median and interquartile ranges (IQR).Student's t-test was to compare differences in normal variables between two independent groups and Mann-Whitney U test for nonnormal variables.Pearson's test was used to assess correlation analyses.Crude ORs calculation and 95% CI have been applied in single SNP analyses and Bonferroni correction for multiple SNPs comparisons has been utilized.

Table 3 .
Loadings of Principal Components in the whole cohort (n = 230).In bold the main loadings exceeding the absolute cut-off value > 0.30.

Table 4 .
Principal Component regression analysis in the whole cohort (n = 230).In bold significant P values.